Membrane proteins represent approximately 30% of the proteome in both prokaryotes and eukaryotes (Wallin and von Heijne, 1998). One of the important steps in membrane protein kinesis is trafficking to the correct site of action. Abnormal protein folding and trafficking is associated with a growing number of diseases (Thomas et al., 1995, Dobson, 2001, Radford et al., 1999). Prominent biogenesis steps include two transport events: anterograde transport and retrograde transport. The former represents a forward progression of vesicle movement toward the cell surface. The latter represents a process of reversal—vesicular trafficking to retrieve selectively marked proteins to the ER (Bonifacino and Glick, 2004).
Due to sequential folding steps and “quality” checks, even slight changes in protein structure or oligomeric state may be detected and, therefore, prevent trafficking to the site of action. The exit from ER to cell surface takes place when the proteins are appropriately assembled. A cell-surface protein contains a “zipcode” that uniquely tags for cell-surface expression. Improper folding or incomplete subunit assembly exposes a molecular feature (structure or sequence) that is recognized by the retention/retrieval machinery. Because little is known about the surface expression “zipcode,” either in terms of signal motifs or of the molecular mechanism, the working of these two opposing forces, ER localization vs. surface localization, in mediating membrane protein expression requires further investigation.
Expression of membrane receptors on the cell surface is a highly regulated event. Signal motifs have been identified that are designed to confer spatial localization of newly synthesized proteins to ER for their final activity or to a transitional compartment before assembly with other proteins and trafficking to the cell surface (Mellman and Warren, 2000). Specific sequences, such as KKXX and RXR motifs, have been identified for their roles in conferring ER localization of membrane proteins (Nilsson et al., 1989; Zerangue et al., 1999). Signal motifs that promote surface expression of membrane proteins have also been identified, including DXE (Nishimura and Balch, 1997) and FCYENE (SEQ ID NO: 249) (Ma et al., 2001). These motifs are thought to function at the step of ER exit.
The existing evidence supports the notion that the opposing forces of ER exit (leading to surface expression) and retrieval (conferring ER localization) operate in two sequential biogenesis steps. Consistent with this view, the retrograde transport, a later step than the ER exit step, is naturally dominant. The selective exit from retrograde transport that allows for protein to progress to the cell surface may be achieved by at least three mechanisms. First, the ER localization signals, such as dibasic and RKR motifs, may be masked as a result of macromolecular assembly of multiple subunits. Second, in addition to the physical masking, the ER localization signals are sensitive to the proximity to cytoplasmic leaflets of membrane. Hence, the ER localization efficiency could be modified by re-positioning a signal into a different zone as a result of protein-protein interaction or folding. The third mechanism is to recruit modular proteins which bind competitively with COPI, the retrograde transport machinery.
To explore possible signal motifs and regulatory pathways of exiting from retrograde transport, a genetic screen was designed to evaluate random peptide sequences for their ability to override ER localization activity and to direct surface expression.